Agent for restoring visual function or agent for preventing deterioration in visual function

ABSTRACT

The purpose of the present invention is to provide an agent for restoring a visual function or an agent for preventing the deterioration in a visual function, which has an excellent visual function restoring ability. The agent for regenerating a visual function or the agent for preventing the deterioration in a visual function according to the present invention contains, as an active ingredient, a chimeric protein having both an amino acid sequence for a microorganism-origin ion-transporting receptor rhodopsin and an amino acid sequence for an animal-origin G-protein-coupled receptor rhodopsin. The chimeric protein is preferably one in which an amino acid sequence for a cytoplasm-side second loop and/or a cytoplasm-side third loop in the amino acid sequence for the microorganism-origin ion-transporting receptor rhodopsin is substituted by an amino acid sequence for a cytoplasm-side second loop and/or a cytoplasm-side third loop in the G-protein-coupled receptor rhodopsin.

TECHNICAL FIELD

The present invention relates to an agent for restoring visual functionor agent for preventing deterioration in visual function.

BACKGROUND ART

Rhodopsin is a photosensitive receptor with a seven transmembranestructure in the retina of humans and animals. Ion channel and ion pumptype rhodopsins derived from microorganisms are also known.

For example, Non Patent Literature 1 discloses an ion channel typerhodopsin, channelrhodopsin 2 (ChR2). Further, Non Patent Literature 2has reported that a certain visual function is restored in mice/rats byintroducing a mutant channelrhodopsin into retinal ganglion cells.

CITATION LIST Non Patent Literature

-   [NPL 1] Bi et al., “Ectopic expression of a microbial-type rhodopsin    restores visual responses in mice with photoreceptor degeneration.”    Neuron. 2006; 50(1): 23-33-   [NPL 2] Tomita et al., “Restoration of the Majority of the Visual    Spectrum by Using Modified Volvox Channelrhodopsin-1”, Molecular    Therapy (2014); 22 8, 1434-1440

SUMMARY OF INVENTION Technical Problem

However, the effect of restoring visual function of ion channel typerhodopsins is still not considered sufficient, such that there is roomfor improvement.

The present invention has been conceived in view of the abovecircumstances. The objective of the invention is to provide an agent forrestoring visual function or agent for preventing deterioration invisual function with an excellent capability to restore visual function.

Solution to Problem

The inventors have found that a chimeric protein prepared by fusing twocompletely different rhodopsins, i.e., a microorganism derived iontransport rhodopsin and an animal derived G protein-coupled receptorrhodopsin in fact has an excellent capability to restore visual functionto complete the present invention. More specifically, the presentinventions are composed of the following configurations.

(1) An agent for restoring visual function or agent for preventingdeterioration in visual function comprising, as an active ingredient, achimeric protein having an amino acid sequence of a microorganismderived ion transport receptor rhodopsin and an amino acid sequence ofan animal derived G protein-coupled receptor rhodopsin.(2) The agent for restoring visual function or agent for preventingdeterioration in visual function of (1), wherein the chimeric proteinhas an amino acid sequence of a second loop on a cytoplasm side and/or athird loop on a cytoplasm side of the amino acid sequence of themicroorganism derived ion transport receptor rhodopsin, replaced with anamino acid sequence of a second loop on a cytoplasm side and/or a thirdloop on a cytoplasm side of the G protein-coupled receptor rhodopsin.(3) The agent for restoring visual function or agent for preventingdeterioration in visual function of (1) or (2), wherein themicroorganism derived ion transport receptor rhodopsin is a rhodopsinderived from a microorganism of the Gloeobacter genus, and the Gprotein-coupled receptor rhodopsin is a bovine or human derivedrhodopsin.(4) The agent for restoring visual function or agent for preventingdeterioration in visual function of any one of (1) to (3), wherein thechimeric protein has an amino acid sequence encoded by a DNA of any oneof the following (a) to (d):(a) a DNA having a base sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4;(b) a DNA having a base sequence that can hybridize under a stringentcondition with a base sequence complementary to a base sequence encodingthe amino acid sequence of any one of SEQ ID NOs: 1 to 4;(c) a DNA having a base sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4 with one or more amino acid substitutions,deletions, and/or additions, and having a visual function restoringcapability or visual function deterioration preventing capability; and(d) a DNA consisting of a base sequence encoding an amino acid sequencehaving 90% or greater homology with the amino acid sequence of any oneof SEQ ID NOs: 1 to 4 and having a visual function restoring capabilityor visual function deterioration preventing capability.(5) An agent for restoring visual function or agent for preventingdeterioration in visual function comprising, as an active ingredient, anexpression vector into which a DNA encoding the amino acid sequence ofthe chimeric protein of any one of (1) to (4) is incorporated.(6) The agent for restoring visual function or agent for preventingdeterioration in visual function of any one of (1) to (5) for use intreating or preventing retinitis pigmentosa.(7) An adeno-associated virus (AAV) vector or lentivirus vector, towhich a sequence of a chimeric protein having an amino acid sequence ofa microorganism derived ion transport receptor rhodopsin and an aminoacid sequence of an animal derived G protein-coupled receptor rhodopsinis inserted.(8) Use of an adeno-associated virus (AAV) vector or lentivirus vector,to which a sequence of a chimeric protein having an amino acid sequenceof a microorganism derived ion transport receptor rhodopsin and an aminoacid sequence of an animal derived G protein-coupled receptor rhodopsinis inserted, for the manufacture of a medicament for restoring visualfunction or preventing deterioration in visual function.

Advantageous Effects of Invention

The present invention can attain an excellent capability to restorevisual function.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is an image of a retina of a wild-type mouse injected withAAV2-CAGGS-EGFP-WPRE-pA into the vitreous body observed under afluorescence microscope.

FIG. 2 is (a) a graph of a result of recording extracellular potentialof retinal ganglion cells by a multielectrode array (MEA) for a controlretinitis pigmentosa model (rdl) mouse, and (b) a graph of a result ofrecording extracellular potential of retinal ganglion cells by MEA for aretinitis pigmentosa model (rdl) mouse injected withAAV2-CAGGS-GR/BvRh-WPRE-pA.

FIG. 3 is a diagram showing a result of recording extracellularpotential of retinal ganglion cells by a multielectrode array (MEA) for(a) a control retinitis pigmentosa model (rdl) mouse and (b) a retinitispigmentosa model (rdl) mouse injected with AAV2-CAGGS-GR/BvRh-WPRE-pA.The top row of FIG. 3 displays a raster plot for 10 firings of retinalganglion cells, and the bottom row of FIG. 3 is a histogram representingthe frequency of firings per second on the vertical axis.

DESCRIPTION OF EMBODIMENTS

While the specific embodiments of the invention are described indetailed hereinafter, the present invention is not limited in any mannerto the following embodiments. The present invention can be practiced byapplying an appropriate modification within the scope of the objects ofthe invention. Explanation is omitted when appropriate for portionswhere a description would be redundant, but such an omission does notlimit the gist of the invention.

<Agent for Restoring Visual Function or Agent for PreventingDeterioration in Visual Function>

The agent for restoring visual function or agent for preventingdeterioration in visual function of the invention comprises, as anactive ingredient, a chimeric protein having an amino acid sequence of amicroorganism derived ion transport receptor rhodopsin and an amino acidsequence of an animal derived G protein-coupled receptor rhodopsin.

Rhodopsin has a pigment called retinal inside, which is activated byreceiving light to transmit a visual signal to the brain. Microorganismderived ion transport receptor rhodopsins can be repeatedly activated byabsorbing light because they do not release retinal by lightirradiation, but are unable to activate a G protein as in animal derivedG protein-coupled receptor rhodopsins. Meanwhile, according to thepresent invention, high activity through the endogenous G protein due tothe G protein-coupled receptor rhodopsin while retaining the function ofrepeated activation of the microorganism derived ion transportreceptor/ion channel type receptor rhodopsin can be attained by fusingan animal derived G protein-coupled receptor rhodopsin to amicroorganism derived ion transport receptor rhodopsin that can berepeatedly used. Such a fusion rhodopsin is expected to attain anexcellent visual restoring effect. In this manner, microorganism derivedrhodopsins and animal derived G protein-coupled receptors are receptorswith completely different functions. The inventors have actually foundthat a chimeric protein combining two such receptors has an excellentcapability to restore a visual function. Since such a chimeric proteincan be repeatedly activated while having high activity as discussedabove, an effect of preventing the deterioration in visual function(e.g., suppressing the progression of retinal diseases such as retinitispigmentosa) is also expected.

Examples of ion transport receptor rhodopsins include ion pump typereceptor rhodopsins and ion channel type receptor rhodopsins.

The chimeric protein of the invention is a chimeric protein of amicroorganism derived ion transport receptor rhodopsin and a Gprotein-coupled receptor rhodopsin, having a seven transmembranestructure. It is preferable in the present invention that a chimericprotein of a microorganism derived ion transport receptor rhodopsin anda G protein-coupled receptor rhodopsin is designed to have both highlevel of function for repeatedly activating the microorganism derivedion transport receptor rhodopsin and G protein activity due to the Gprotein-coupled receptor rhodopsin. From this viewpoint, it ispreferable that the chimeric protein of the invention has an amino acidsequence of a second loop on a cytoplasm side and/or a third loop on acytoplasm side of the amino acid sequence of the microorganism derivedion transport receptor rhodopsin substituted with an amino acid sequenceof a second loop on a cytoplasm side and/or a third loop on a cytoplasmside of the G protein-coupled receptor rhodopsin, because bothactivities are maintained high and especially because high visualfunction restoring capability is attained. The “second loop on acytoplasm side” and “third loop on a cytoplasm side” refer to loops atposition 2 from the N-terminus side and position 3 from the N-terminusside among the seven loops, respectively.

Examples of microorganism derived ion transport receptor rhodopsinsinclude rhodopsins derived from microorganisms, e.g., belonging toeubacteria such as the Gloeobacter genus and the like, eukaryotes suchas the Volvox genus, Chlamydomonas genus, Guillardia genus, and thelike. Examples of the Gloeobacter genus include Gloeobacter violaceusand the like. Examples of the Volvox genus include Volvox carteri andthe like. Examples of the Chlamydomonas genus include Chlamydomonasreinhardtii and the like. Examples of Guillardia genus includeGuillardia theta and the like. Conformational compatibility with a Gprotein activation loop and the membrane translocation efficiency areconsidered important for attaining a higher visualrestoration/prophylactic effect. Microorganism derived ion transportreceptor rhodopsins are thus preferably of the Gloeobacter genus due tothe especially excellent conformational compatibility with a G proteinactivation loop and membrane translocation efficiency. Gloeobacterviolaceus is especially preferable among microorganisms of theGloeobacter genus. It is also preferable to combine and fuse those of amicroorganism of the Gloeobacter genus with a bovine or human derived Gprotein-coupled receptor rhodopsin among animal derived Gprotein-coupled receptor rhodopsins. The Gloeobacter genus is alsopreferable in terms of having an important property of being expressedwell in E. coli, which is a eubacterium, and human cells, which areeukaryotes.

Examples of animal derived G protein-coupled receptor rhodopsins includerhodopsins derived from a cow, human, mouse, rat, cat, dog, swine,sheep, horse, or the like. Among them, bovine and human derivedrhodopsins are particularly preferable.

More specifically, a chimeric protein preferably has an amino acidsequence encoding the DNA of any one of the following (a) to (d):

(a) a DNA having a base sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4;(b) a DNA having a base sequence that can hybridize under a stringentcondition with a base sequence complementary to a base sequence encodingthe amino acid sequence of any one of SEQ ID NOs: 1 to 4;(c) a DNA having a base sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4 with one or more amino acid substitutions,deletions, and/or additions, and having a visual function restoringcapability or visual function deterioration preventing capability; and(d) a DNA consisting of a base sequence encoding an amino acid sequencehaving 90% or greater homology with the amino acid sequence of any oneof SEQ ID NOs: 1 to 4 and having a visual function restoring capabilityor visual function deterioration preventing capability.

The second loop on the cytoplasm side of the G protein-coupled receptorrhodopsin discussed above preferably has an amino acid encoding the DNAof the following (e) to (h):

(e) a DNA having a base sequence encoding the amino acid sequence of SEQID NO: 5 or 6;(f) a DNA having a base sequence that can hybridize under a stringentcondition with a base sequence complementary to a base sequence encodingthe amino acid sequence of SEQ ID NO: 5 or 6;(g) a DNA having a base sequence encoding the amino acid sequence of SEQID NO: 5 or 6 with one or more amino acid substitutions, deletions,and/or additions; and(h) a DNA consisting of a base sequence encoding an amino acid sequencehaving 90% or greater homology with the amino acid sequence of SEQ IDNO: 5 or 6.

The third loop on the cytoplasm side of the G protein-coupled receptorrhodopsin discussed above preferably has an amino acid encoding the DNAof the following (i) to (l):

(i) a DNA having a base sequence encoding the amino acid sequence of SEQID NO: 7;(j) a DNA having a base sequence that can hybridize under a stringentcondition with a base sequence complementary to a base sequence encodingthe amino acid sequence of SEQ ID NO: 7;(k) a DNA having a base sequence encoding the amino acid sequence of SEQID NO: 7 with one or more amino acid substitutions, deletions, and/oradditions; and(l) a DNA consisting of a base sequence encoding an amino acid sequencehaving 90% or greater homology with the amino acid sequence of SEQ IDNO: 7.

A base sequence encoding the amino acid sequence of any one of SEQ IDNOs: 1 to 4 is a preferred sequence of a base sequence encoding thechimeric protein of the invention. The base sequence encoding the aminoacid sequence of any one of SEQ ID NOs: 1 to 4 has a visual functionrestoring capability or visual function deterioration preventingcapability. As used herein, “base sequence has a visual functionrestoring capability or visual function deterioration preventingcapability means that a polypeptide encoded by the base sequence has avisual function restoring capability or visual function deteriorationpreventing capability. A DNA having a base sequence encoding the aminoacid sequence of any one of SEQ ID NOs: 1 to 4 further encompassesvarious mutants and homologs having a visual function restoringcapability or visual function deterioration preventing capability.Mutants and homologs of a DNA having a base sequence encoding the aminoacid sequence of any one of SEQ ID NOs: 1 to 4 encompass, for example,DNAs having a base sequence that can hybridize under a stringentcondition with a base sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4. Further, mutants and homologs of a DNA havinga base sequence encoding the amino acid sequence of any one of SEQ IDNOs: 5 to 7 encompass DNAs having a base sequence that can hybridizeunder a stringent condition with a base sequence encoding the amino acidsequence of any one of SEQ ID NOs: 5 to 7. Examples of “stringentcondition” include conditions for performing a reaction at 40 to 70° C.(preferably 50 to 67° C. and more preferably 60 to 65° C.) in a normalhybridization buffer and washing in a detergent with a saltconcentration of 15 to 300 mM (preferably 15 to 150 mM, more preferably15 to 60 mM, and still more preferably 30 to 50 mM).

Any one of SEQ ID NOs: 1 to 4 can be used as the amino acid sequence ofthe chimeric protein of the invention. A DNA encoding the amino acidsequence of the chimeric protein of the invention encompasses DNAshaving a base sequence encoding the amino acid sequence of any one ofSEQ ID NOs: 1 to 4 with one or more amino acid substitutions, deletions,and/or additions. In this regard, “one or more” in any one of SEQ IDNOs: 1 to 4 is generally 50 amino acids or less, preferably 30 aminoacids or less, and still more preferably 10 amino acids or less (e.g., 5amino acids or less, 3 amino acids or less, or one amino acid). Further,“one or more” in any one of SEQ ID NOs: 5 to 7 is generally 6 aminoacids or less, preferably 5 amino acids or less, and still morepreferably 4 amino acids or less (e.g., 3 amino acids or less, 2 aminoacids or less, and one amino acid). When maintaining a visual functionrestoring capability or visual function deterioration preventingcapability of a chimeric protein, it is desirable that an amino acidresidue to be mutated is mutated to another amino acid which conservesthe property of an amino acid side chain. Examples of properties of anamino acid side chain include hydrophobic amino acids (A, I, L, M, F, P,W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T),amino acids with an aliphatic side chain (G, A, V, L, I, P), amino acidswith a hydroxyl group containing side chain (S, T, Y), amino acids witha sulfur atom containing side chain (C, M), amino acids with acarboxylic acid and amide containing side chain (D, N, E, Q), aminoacids with a base containing side chain (R, K, H), and amino acids withan aromatic containing side chain (H, F, Y, W) (each symbol within theparenthesis represents the one-letter code of an amino acid). It isknown that proteins having an amino acid sequence modified by one ormore amino acid residue deletions, additions, and/or substitutions withanother amino acid to the amino acid sequence maintain the biologicalactivity thereof (Mark, D. F. et al., Proc. Natl. Acad. Sci. USA (1984)81, 5662-5666, Zoller, M. J. & Smith, M. Nucleic Acids Research (1982)10, 6487-6500, Wang, A. et al., Science 224, 1431-1433,Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79,6409-6413).

Mutants and homologs of a DNA having a base sequence encoding the aminoacid sequence of any one of SEQ ID NOs: 1 to 4 encompass DNAs consistingof a base sequence having high homology with a base sequence encodingthe amino acid sequence of any one of SEQ ID NOs: 1 to 4. Such a DNApreferably has homology of 90% or greater, and still more preferably 95%or greater (96% or greater, 97% or greater, 98% or greater, or 99% orgreater) with a base sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4. Mutants and homologs of a DNA having a basesequence encoding the amino acid sequence of any one of SEQ ID NOs: 5 to7 encompass DNAs consisting of a base sequence having high homology witha base sequence encoding the amino acid sequence of any one of SEQ IDNOs: 5 to 7. Such a DNA preferably has homology of 90% or greater, andstill more preferably 95% or greater (96% or greater, 97% or greater,98% or greater, or 99% or greater) with a base sequence encoding theamino acid sequence of any one of SEQ ID NOs: 5 to 7. The homology ofamino acid sequences and base sequences can be determined by thealgorithm BLAST developed by Karlin and Altschul (Proc. Natl. Acad. Sci.USA 90: 5873-5877, 1993). Programs called BLASTN and BLASTX have beendeveloped based on this algorithm (Altschul et al. J. Mol. Biol. 215:403-410, 1990). When analyzing a base sequence using BLASTN based onBLAST, parameters are set to, for example, score=100 and wordlength=12.When analyzing an amino acid sequence using BLASTX based on BLAST,parameters are set to, for example, score=50 and wordlength=3. Whenusing BLAST and Gapped BLAST programs, the default parameters of eachprogram are used. The specific approaches of these analysis methods areknown (http://www.ncbi.nlm.nih.gov).

As used herein, “DNA” may be a sense strand or an antisense strand(e.g., can be used as a probe). The shape thereof may be a single strandor double strand. DNA may also be genomic DNA, cDNA, or synthesized DNA.

The method of obtaining DNA of the invention is not particularlylimited. Examples thereof include known methods such as a method ofobtaining cDNA by reverse transcription from mRNA (e.g., RT-PCR method),method of adjusting from genomic DNA, method of synthesizing by chemicalsynthesis, and method of isolating from a genomic DNA library or a cDNAlibrary (see, for example, Japanese Laid-Open Publication No. 11-29599).

A chimeric protein used in the agent for restoring visual function oragent for preventing deterioration in visual function of the inventioncan be prepared, for example, by using a transformant introduced with anexpression vector comprising a DNA encoding the aforementioned chimericprotein. For example, the transformant is first cultured under suitableconditions to synthesize a chimeric protein encoded by the DNA. Thesynthesized protein can then be retrieved from the transformant orculture to obtain the chimeric protein of the invention.

More specifically, this can be made by inserting a DNA encoding theaforementioned chimeric protein into a suitable expression vector. The“suitable vector” may be any vector that can be replicated and retainedor self-proliferate within various hosts of prokaryotes and/oreukaryotes. The vector can be appropriately selected depending on theobjects of use. For obtaining a large quantity of DNA, a high copynumber vector, for example, can be selected. For obtaining a polypeptide(chimeric protein), an expression vector can be selected. Specificexamples of vectors include, but are not particularly limited to, knownvectors described in Japanese Laid-Open Publication No. 11-29599.

Expression vectors can not only synthesize a chimeric protein, but alsobe used in the agent for restoring visual function or agent forpreventing deterioration in visual function of the invention. In otherwords, the agent for restoring visual function or agent for preventingdeterioration in visual function of the invention may comprise, as anactive ingredient, an expression vector into which a DNA encoding theamino acid sequence of the aforementioned chimeric protein isincorporated. Such an expression vector can be used in restoring visualfunction or prevention of deterioration in visual function by directintroduction into a human. As a vector in such use, a vector that can beintroduced into a human cell is used. Preferred examples of such avector include adeno-associated virus vectors (AAV vectors) andlentivirus vectors.

A method of introducing a vector can be appropriately selected dependingon the type of host or vector or the like. Specific examples of themethod include, but are not particularly limited to, known methods suchas the protoplast and competent methods when bacteria are used as thehost (see, for example, Japanese Laid-Open Publication No. 11-29599).When an expression vector is used as an active ingredient of the agentfor restoring visual function or agent for preventing deterioration invisual function of the invention, the aforementioned AAV vector or thelike can be introduced, for example, by injection into the eye.

A host to which an expression vector may be any host that is compatiblewith the expression vector and can be transformed. Specific examples ofthe host include, but are not particularly limited to, knownnaturally-occurring or artificially established cells such as bacteria,yeast, animal cells, and insect cells (see Japanese Laid-OpenPublication No. 11-29599) and animals such as humans and mice. Atransformant can be cultured by suitably selecting a medium from knownnutrient media depending on the type of the transformant or the like andappropriately adjusting the temperature, pH of the nutrient medium,culture time, and the like, so that a chimeric protein can be readilyobtained in large quantities (see, for example, Japanese Laid-OpenPublication No. 11-29599).

An isolation method and purification method of a chimeric protein is notparticularly limited. Examples thereof include known methods such asmethods of utilizing solubility, methods utilizing the difference inmolecular weights, and methods utilizing charges (see, for example,Japanese Laid-Open Publication No. 11-29599).

As used herein, “active ingredient” refers to an ingredient contained atan amount needed to attain the effect of restoring visual function orthe effect of preventing deterioration in visual function. Otheringredients may also be contained, as long as the effect is not reducedbelow a desired level. The agent for restoring visual function or agentfor preventing deterioration in visual function of the invention mayalso be formulated. Further, the route of administration of the agentfor restoring visual function or agent for preventing deterioration invisual function of the invention may be either oral or parenteral. Theroute of administration can be appropriately determined depending on theform of formulation or the like.

For oral administration, the agent may be formulated into various formssuch as tablets, granules, fine granules, powder, and capsules for use.An additive commonly used in a formulation such as a binding agent,covering agent, excipient, lubricant, disintegrant, or humectant mayalso be included. In addition thereto, formulations for oraladministration may be formulated as a liquid formulation such as anaqueous solution for internal use, suspension, emulsion, or syrup. Theformulation may also be formulated as a dry formulation that isdissolved in a solvent upon use.

For parenteral administration, the agent may be formulated to becontained in a unit dose ampule or multidose container or tube. Anadditive such as a stabilizer, buffer, preservative, or isotonizingagent may also be included. A formulation for parenteral administrationmay also be formulated into a powder form that can be dissolved in asuitable carrier (sterilized water or the like) upon use.

Examples of parenteral administration include intravitrealadministration, subconjunctival administration, intra-anterior chamberadministration, and eye drops, and intravitreal administration ispreferred.

The agent for restoring visual function or agent for preventingdeterioration in visual function of the invention discussed above can beused for restoring visual function or preventing deterioration in visualfunction by administration to humans using the aforementioned method.

As use herein, “visual function restoration” refers to improvement ofdeteriorated visual function, which may be a partial or completerestoration of the visual function. Further, “prevention ofdeterioration in visual function” refers to prevention of deteriorationin visual function, suppression of progression in deterioration ofvisual function, and the like. Examples of such visual function includevision, contrast sensitivity, light adaptation, color perception, andthe like.

The agent for restoring visual function or agent for preventingdeterioration in visual function of the invention may be used inapplications expected from restoration of visual function or preventionof deterioration in visual function. For example, the agent may be usedin treating or preventing a disease associated with deterioration invisual function. Examples of diseases associated with deterioration invisual function include retinitis pigmentosa, age related maculardegeneration, myopic maculopathy, macular dystrophy, diabeticretinopathy, uveitis, retinal detachment, and the like.

<Vector>

The present invention includes adeno-associated virus (AAV) vectors andlentivirus vectors, to which a sequence of a chimeric protein having anamino acid sequence of a microorganism derived ion transport receptorrhodopsin and an amino acid sequence of an animal derived Gprotein-coupled receptor rhodopsin is inserted.

The present invention also includes the use of an adeno-associated virus(AAV) vector or lentivirus vector, to which a sequence of a chimericprotein having an amino acid sequence of a microorganism derived iontransport receptor rhodopsin and an amino acid sequence of an animalderived G protein-coupled receptor rhodopsin is inserted, for themanufacture of a medicament for restoring visual function or preventingdeterioration in visual function.

The same chimeric protein discussed above can be used as the chimericprotein.

EXAMPLES

Experiments related to visual function were conducted using mice asdescribed below.

(Experimental Animal)

For the experiments, wild-type mouse (C57BL/6J, CLEA Japan Inc.) andretinitis pigmentosa model (rdl) mouse (C3H/HeJ Jcls, CLEA Japan Inc.)were used, which were both 3-week old male.

(Production of DNA encoding chimeric protein (GR/BvRh) A DNA encoding achimeric protein, in which a sequence corresponding to 137th to 145thamino acids from the N-terminus corresponding to the second loop on thecytoplasm side of Gloeobacter violaceus Rhodopsin ((GR), SEQ ID NO: 8)was replaced with a sequence corresponding to 137th to 145th amino acidsof a bovine rhodopsin (BvRh) (SEQ ID NO: 9), and a sequencecorresponding to 198th to 206th amino acids from the N-terminuscorresponding to the third loop on the cytoplasm side of Gloeobacterviolaceus Rhodopsin was replaced with a sequence corresponding to 225thto 252th amino acids of the bovine rhodopsin, and the 132nd amino acid,glutamic acid, of Gloeobacter violaceus Rhodopsin was replaced withglutamine, was inserted into a pCDNA3.1 vector. The mutant was producedby the QuicChange method.

(Production of Adeno-Associated Virus (AAV) Vector to which a Sequenceof Chimeric Protein is Inserted)

An EGFP or GR/BvRh gene was subcloned to an AAV2 shuttle plasmid toproduce AAV2-CAGGS-EGFP-WPRE-pA (vector for expressing EGFP) andAAV2-CAGGS-GR/BvRh-WPRE-pA (vector for expressing a chimeric protein) asvirus expressing constructs. Viral vectors were packaged by transfectionof three types of plasmids, i.e., vector plasmid, AAV vector plasmid,and adenovirus helper plasmid, into HEK 293 cells. Cesium chloridemethod was used for the purification of the viral vectors. In thevectors, “ITR” is an abbreviation for “Inverted Terminal Repeat”.“CAGGS” is a sequence of a region of a CAG promoter. “WPRE” is anabbreviation for “woodchuck hepatitis virus post-transcriptionalregulatory element”. “pA” refers to a peptide tag. “EGFP” is anabbreviation for “enhanced green fluorescent protein”.

(Vitreous Body Injection)

A mixture of medetomidine hydrochloride (0.75 mg/kg), midazolam (4mg/kg), butorphanol tartrate (5 mg/kg) was intraperitoneallyadministered to a wild-type mouse or retinitis pigmentosa model (rdl)mouse. Under systemic anesthesia, a microsyringe equipped with a 32gauge needle was used to inject the aforementioned AAV vector(“AAV2-CAGGS-EGFP-WPRE-pA” or “AAV2-CAGGS-GR/BvRh-WPRE-pA”) at 1×10¹²vg/ml and 1 μl, respectively, into the vitreous body from near the or aserrata.

(Reporter Observation)

The retina was extracted from a wild-type mouse injected withAAV2-CAGGS-EGFP-WPRE-pA after 7 weeks from injection and immobilized for1 hour with 4% paraformaldehyde. The whole-mounted retina was observedunder a fluorescence microscope. FIG. 1 shows the result thereof. InFIG. 1, GCL means the ganglion cell layer, INL means the inner nuclearlayer, and ONL means the outer nuclear layer. Green fluorescence (e.g.,arrow in FIG. 1) was observed in the retina as a result of observation.Thus, it was possible to confirm that vector introduction and expressionof a gene of interest were normal.

(Multielectrode Array Recording (MEA))

An eye ball was extracted under general anesthesia after 7 weeks frominjecting AAV2-CAGGS-GR/BvRh-WPRE-pA to a retinitis pigmentosa model(rdl) mouse. The eye ball was then left standing in an Ames medium(Sigma-Aldrich, St Louis, Mo.; A1420) bubbled with 95% O₂ and 5% CO₂,then the retina was extracted. The retina was mounted so that theganglion cell layer contacted an electrode facing down, and subjected tolight stimulation (white light, 1000 cd/m², 1 second) to recordextracellular potential of retinal ganglion cells. Extracellularpotential of retinal ganglion cells was also recorded by the same methodusing a retinitis pigmentosa model (rdl) mouse which had not beeninjected with AAV2-CAGGS-GR/BvRh-WPRE-pA as a control. A MEA2100-Litesystem (Multi-Channel Systems, Reutlingen, Germany) was used for themultielectrode array recording. FIG. 2 shows the results thereof. FIG.2(a) shows a graph for the control mouse, and FIG. 2(b) shows a graphfor a mouse injected with AAV2-CAGGS-GR/BvRh-WPRE-pA. In the graphs ofFIG. 2, the horizontal axis indicates the time elapsed, and the regionsindicated by an arrow indicate regions where light stimulation wasapplied.

As shown in FIG. 2, no change was observed in the region where lightstimulation was applied for the control, but it was found that thepotential increased for the mouse injected withAAV2-CAGGS-GR/BvRh-WPRE-pA. In view of these results, it was found thatGR/BvRh has an effect of restoring visual function against retinitispigmentosa.

Further, multielectrode array recording was performed by the sameapproach as above to obtain 10 firings of retinal ganglion cellsdisplayed in a raster plot (top row of FIG. 3), and histogramsrepresenting the frequency of firings per second on the vertical axis(bottom row of FIG. 3). Light stimulation was applied from 0 to 1second. FIG. 3(a) shows a graph for a control mouse, and FIG. 3(b) showsa graph for a mouse injected with AAV2-CAGGS-GR/BvRh-WPRE-pA.

As shown in FIG. 3, a photoresponse was not observed in the controlmouse, whereas firing of ganglion cells was observed in the mouseinjected with AAV2-CAGGS-GR/BvRh-WPRE-pA, so that a visual restorationeffect was observed.

1.-8. (canceled)
 9. A method for treating or preventing a retinaldisease in a subject in need therefor or at risk, comprising the step ofadministering to said subject an effective amount of a nucleic acidmolecule comprising a nucleic acid sequence, wherein the nucleic acidsequence encodes a chimeric protein comprising an amino acid sequence ofan ion transport receptor rhodopsin derived from a microorganism of theGloeobacter genus and an amino acid sequence of a bovine or humanderived G protein-coupled receptor rhodopsin, wherein the chimericprotein comprises an amino acid sequence of at least one of a secondloop on the cytoplasm side and a third loop on the cytoplasm side of theamino acid sequence of the microorganism derived ion transport receptorrhodopsin substituted with an amino acid sequence of at least one of asecond loop on the cytoplasm side and a third loop on the cytoplasm sideof the G protein-coupled receptor rhodopsin.
 10. A method for treatingor preventing retinitis pigmentosa, age related macular degeneration,myopic maculopathy, macular dystrophy, diabetic retinopathy, uveitis orretinal detachment in a subject in need therefor or at risk, comprisingthe step of administering to said subject an effective amount of anucleic acid molecule comprising a nucleic acid sequence, wherein thenucleic acid sequence encodes a chimeric protein comprising an aminoacid sequence of an ion transport receptor rhodopsin derived from amicroorganism of the Gloeobacter genus and an amino acid sequence of a Gprotein-coupled receptor rhodopsin derived from bovine or human, whereinthe chimeric protein comprises an amino acid sequence of at least one ofa second loop on the cytoplasm side and a third loop on the cytoplasmside of the amino acid sequence of the microorganism derived iontransport receptor rhodopsin substituted with an amino acid sequence ofat least one of a second loop on the cytoplasm side and a third loop onthe cytoplasm side of the G protein-coupled receptor rhodopsin.
 11. Themethod of claim 9, wherein the retinal disease is selected from thegroup consisting of retinitis pigmentosa, age related maculardegeneration, myopic maculopathy, macular dystrophy, diabeticretinopathy, and retinal detachment.
 12. The method of claim 9, whereinthe retinal disease is retinitis pigmentosa.
 13. The method of claim 9,wherein the nucleic acid molecule comprises a nucleic acid sequenceaccording to any one of the following (a) to (d): (a) a nucleic acidsequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to4; (b) a nucleic acid sequence that hybridizes under stringentconditions with a nucleic acid sequence complementary to a nucleic acidsequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to4; (c) a nucleic acid sequence encoding the amino acid sequence of anyone of SEQ ID NOs: 1 to 4 comprising at least one or more amino acidsubstitutions, deletions, and additions, and having a visual functionrestoring capability or visual function deterioration preventingcapability; and (d) a nucleic acid sequence encoding an amino acidsequence having 90% or greater homology with the amino acid sequence ofany one of SEQ ID NOs: 1 to 4 and having a visual function restoringcapability or visual function deterioration preventing capability. 14.The method according to claim 9, wherein the nucleic acid molecule isincorporated in an expression vector.
 15. A method for treating orpreventing a retinal disease in a subject in need therefor or at risk,comprising the step of administering to said subject an effective amountof a chimeric protein comprising an amino acid sequence of an iontransport receptor rhodopsin derived from a microorganism of theGloeobacter genus and an amino acid sequence of a bovine or humanderived G protein-coupled receptor rhodopsin, wherein the chimericprotein comprises an amino acid sequence of at least one of a secondloop on the cytoplasm side and a third loop on the cytoplasm side of theamino acid sequence of the microorganism derived ion transport receptorrhodopsin substituted with an amino acid sequence of at least one of asecond loop on the cytoplasm side and a third loop on the cytoplasm sideof the G protein-coupled receptor rhodopsin.
 16. An adeno-associatedvirus (AAV) vector or lentivirus vector comprising a nucleic acidsequence, wherein the nucleic acid sequence encodes a chimeric proteincomprising an amino acid sequence of an ion transport receptor rhodopsinderived from a microorganism of the Gloeobacter genus and an amino acidsequence of a bovine or human derived G protein-coupled receptorrhodopsin, wherein the chimeric protein comprises an amino acid sequenceof at least one of a second loop on the cytoplasm side and a third loopon the cytoplasm side of the amino acid sequence of the microorganismderived ion transport receptor rhodopsin substituted with an amino acidsequence of at least one of a second loop on the cytoplasm side and athird loop on the cytoplasm side of the G protein-coupled receptorrhodopsin.
 17. A nucleic acid molecule encoding a chimeric protein,wherein the chimeric protein comprises an amino acid sequence of an iontransport receptor rhodopsin derived from a microorganism of theGloeobacter genus and an amino acid sequence of a bovine or humanderived G protein-coupled receptor rhodopsin, wherein the chimericprotein comprises an amino acid sequence of at least one of a secondloop on the cytoplasm side and a third loop on the cytoplasm side of theamino acid sequence of the microorganism derived ion transport receptorrhodopsin substituted with an amino acid sequence of at least one of asecond loop on the cytoplasm side and a third loop on the cytoplasm sideof the G protein-coupled receptor rhodopsin.
 18. A chimeric proteincomprising an amino acid sequence of an ion transport receptor rhodopsinderived from a microorganism of the Gloeobacter genus and an amino acidsequence of a bovine or human derived G protein-coupled receptorrhodopsin, wherein the chimeric protein comprises an amino acid sequenceof at least one of a second loop on the cytoplasm side and a third loopon the cytoplasm side of the amino acid sequence of the microorganismderived ion transport receptor rhodopsin substituted with an amino acidsequence of at least one of a second loop on the cytoplasm side and athird loop on the cytoplasm side of the G protein-coupled receptorrhodopsin.